Ion generation using wetted porous material

ABSTRACT

The invention generally relates to systems and methods for mass spectrometry analysis of samples. In certain embodiments, the invention provides a mass spectrometry probe including at least one porous material connected to a high voltage source, in which the porous material is discrete from a flow of solvent.

RELATED APPLICATIONS

The present invention claims priority to and the benefit of U.S.provisional patent application Ser. No. 61/174,215, filed Apr. 30, 2009,U.S. provisional patent application Ser. No. 61/246,707 filed Sep. 29,2009, and U.S. provisional patent application Ser. No. 61/308,332, filedFeb. 26, 2010, the content of each of which is incorporated by referenceherein in its entirety.

TECHNICAL FIELD

The invention generally relates to systems and methods for massspectrometry analysis of samples.

BACKGROUND

Mass spectrometry is a very sensitive analytical method used forimportant research and for applications of analytical chemistry,particularly life science. Electrospray ionization (ESI) is generallyregarded as the best-characterized and most efficient method forionization of molecules in solution phase. The process can beconveniently divided into three stages: droplet formation, dropletevaporation and ion formation (Gaskell, S. J. Journal of MassSpectrometry 1997, 32, 677-688). When a strong electric field is appliedto a solution flowing through a mass spectrometer probe, a Taylor coneis formed at the tip of the probe, resulting in a mist of small dropletsbeing emitted from the tip of this cone. Due to the evaporation of thefree droplets and Coulombic forces, ions of sample analyte are produced.The ions enter a mass spectrometer and are subsequently analyzed.

A problem with ESI is that sample preparation is still a necessary stepbefore ESI can be used for analysis of many types of samples. Prior toanalyzing a sample by ESI mass spectrometry, the sample will undergoextraction and filtration protocols to purify the sample, for example toremove salts and detergents. Such protocols are complex, time-consuming,and expensive. Further, reagents used during the purification processcan interfere with subsequent analysis of a target analyte in thepurified sample. Additionally, samples that are not in solution must bedissolved as well as purified prior to ESI analysis.

More recently, the concept of ambient ionization has been developed, andnow this family of ambient ionization has more than twenty members, suchas desorption electrospray ionization (DESI) and direct analysis in realtime (DART). Ambient ionization by mass spectrometry allows theionization of analytes under an ambient environment from condensed-phasesamples without much or even any sample preparation and/orpre-separation, offering a solution for real time and in situ analysisfor complex mixtures and biological samples. These ambient ionizationmethods are leading are extending the mass spectrometry revolution inlife science, environment monitoring, forensic applications andtherapeutic analysis. However, the above described ambient ionizationtechniques still require pneumatic assistance, a continuous flow ofsolvent, and a high voltage power supply for the analysis of samples.

There is an unmet need for systems and methods that can combine samplepreparation and pre-treatment and the ionization process for massanalysis of samples that do not require pneumatic assistance or acontinuous flow of solvent for the analysis of the samples.

SUMMARY

The invention generally relates to new systems and methods of generatingions from fluids and solid samples for mass spectrometric analysis.Porous materials, such as filter paper or similar materials are used tohold and transfer liquids, and ions are generated directly from theedges of the materials when a high electric voltage is applied to thematerials. The porous material is kept discrete (i.e., separate ordisconnected from) from a flow of solvent. Instead, a sample is eitherspotted onto the porous material or the porous material is wetted andused to swab a surface containing the sample. The porous material withspotted or swabbed sample is then wetted and connected to a high voltagesource to produce ions of the sample which are subsequently analyzed.The sample is transported through the porous material without the needof a separate solvent flow.

Devices and methods of the invention combine sample preparation andpre-treatment with the ionization process needed for mass analysis ofsamples. Device and methods of the invention allow for rapid and directanalysis of chemicals in raw biological samples of complex matrices,such as biofluids and tissues, without sample preparation. In particularembodiments, devices and methods of the invention allow for the analysisof a dried spots of blood or urine.

An aspect of the invention provides a mass spectrometry probe includinga porous material connected to a high voltage source, in which theporous material is discrete from a flow of solvent. Exemplary porousmaterials include paper, e.g., filter paper, or PVDF membrane. Theporous material can be of any shape. In certain embodiments, the porousmaterial is provided as a triangular piece.

In certain embodiments, the probe further includes a discrete amount ofa solvent, e.g., a droplet or droplets, applied to the porous material.The solvent is applied as a droplet or droplets, and in an amountsufficient to wet the porous material. Once applied to the porousmaterial, the solvent can assist transport of the sample through theporous material. The solvent can contain an internal standard. Thesolvent/substrate combination can allow for differential retention ofsample components with different chemical properties. In certainembodiments, the solvent minimizes salt and matrix effects. In otherembodiments, the solvent includes chemical reagents that allow foron-line chemical derivatization of selected analytes.

Another aspect of the invention provides a system for analyzing a samplematerial including, a probe including a porous material connected to ahigh voltage source, in which the porous material is kept separate froma flow of solvent, and a mass analyzer. The mass analyzer can be that ofa benchtop mass spectrometer or a handheld mass spectrometer. Exemplarymass analyzers include a quadrupole ion trap, a rectilinear ion trap, acylindrical ion trap, a ion cyclotron resonance trap, and an orbitrap.

Another aspect of the invention includes a method for analyzing a sampleincluding, contacting a sample to a porous material, in which the porousmaterial is kept separate from a flow of solvent, applying a highvoltage to the porous material to generate ions of an analyte in thesample that are expelled from the porous material, and analyzing theexpelled ions. The method can further include applying a discreteamount, e.g., a droplet or droplets, of a solvent to the porousmaterial. In certain embodiments, analyzing involves providing a massanalyzer to generate a mass spectrum of analytes in the sample.

In certain embodiments, the sample is a liquid. In other embodiments,the sample is a solid. In embodiments in which the sample is a solid,the porous material can be used to swab the sample from a surface. Asolvent can be applied to the porous material prior to or after thesolid has been swabbed. Exemplary samples include chemical species orbiological species.

Another aspect of the invention provides a method of ionizing a sampleincluding applying a high voltage to a porous material to generate ionsof an analyte in the sample, in which the porous material remainsseparate from a solvent flow. Exemplary porous materials include paperor PVDF membrane.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 panel (A) is a drawing of a sample solution being fed to a pieceof paper for electrospray ionization. FIG. 1 panel (B) is a drawing of asample solution pre-spotted onto the paper and a droplet of solventbeing subsequently supplied to the paper for electrospray ionization.

FIG. 2 panel (A) is a MS spectrum of heroin (concentration: 1 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 2 panel (B) is a MS/MS spectrum of heroin(concentration: 1 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 3 panel (A) is a MS spectrum of caffeine (concentration: 10 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 3 panel (B) is a MS/MS spectrum of caffeine(concentration: 10 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 4 panel (A) is a MS spectrum of benzoylecgonine (concentration: 10ppm, volume: 10 μl solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probesof the invention. FIG. 4 panel (B) is a MS/MS spectrum ofbenzoylecgonine (concentration: 10 ppb, volume: 10 μl, solvent:MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 5 panel (A) is a MS spectrum of serine (concentration: 1 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 5 panel (B) is a MS/MS spectrum of serine(concentration: 100 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 6 panel (A) is a MS spectrum of peptide bradykinin2-9(concentration: 10 ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)) using probes of the invention. FIG. 6 panel (B) is a MS/MSspectrum of bradykinin2-9 (concentration: 1 ppm, volume: 10 μl, solvent:MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 7 panel (A) is a MS/MS spectrum showing that heroin can be detectedfrom whole blood sample by a “spot” method. FIG. 7 panel (B) shows theMS/MS spectrum of the blood spot without heroin.

FIG. 8 panel (A) MS/MS spectrum shows heroin can be detected from rawurine sample by a “spot” method. FIG. 8 panel (B) shows the MS/MSspectrum of the urine spot without heroin.

FIG. 9 panel (A) is a MS spectrum showing the caffeine detected from acola drink without sample preparation. FIG. 9 panel (B) is a MS spectrumshowing caffeine detected from coffee powder. A paper slice was used tocollect the coffee powder from a coffee bag by swabbing the surface.

FIG. 10 shows MS spectra of urine analysis without sample preparation.FIG. 10 panel (A) is a MS spectrum showing that caffeine was detected inurine from a person who consumed coffee. FIG. 10 panel (B) is a MSspectrum showing that caffeine was not detected in urine from a personwho had not consumed any coffee.

FIG. 11 are MS spectra showing the difference between peptide analysis(10 ppm of bradykinin 2-9) on (A) paper triangle and (B) PVDF membraneusing the same parameters (˜2 kV, Solvent: MeOH:H₂O=1:1).

FIG. 12 shows direct MS spectra of plant tissues using sliced tissues offour kinds of plants. (A) Onion, (B) Spring onion, and two differentleaves (C) and (D).

FIG. 13 shows MS/MS spectra of Vitamin C. FIG. 13 panel (A) directanalysis of onion without sample preparation. FIG. 13 panel (B) usingstandard solution.

FIG. 14 panel A is a picture showing dried blood spot analysis on paper;0.4 μL of whole blood is applied directly to a triangular section ofchromatography paper (typically height 10 mm, base 5 mm). A copper clipholds the paper section in front of the inlet of an LTQ massspectrometer (Thermo Fisher Scientific, San Jose, Calif.) and a DCvoltage (4.5 kV) is applied to the paper wetted with 10 μLmethanol/water (1:1 v/v). Panel B shows the molecular structure ofimatinib (GLEEVEC) and paper spray tandem mass spectrum of 0.4 μL wholeblood containing 4 μg/mL imatinib. Imatinib is identified and quantified(inset) by the MS/MS transition m/z 494→m/z 394 (inset). Panel C shows aquantitative analysis of whole blood spiked with imatinib (62.5−4 μg/mL)and its isotopomers imatinib-d8 (1 μg/mL). Inset plot shows lowconcentration range.

FIG. 15 is a paper spray mass spectrum of angiotensin I solution. Theinset shows an expanded view over the mass range 630-700.

FIG. 16 is a mass spectrum showing direct analysis of hormones in animaltissue by probes of the invention.

FIG. 17 panels A and B are mass spectra showing direct analysis of humanprostate tumor tissue and normal tissue.

FIG. 18 is a mass spectrum of whole blood spiked with 10 μg/mL atenolol.The data was obtained by combining systems and methods of the inventionwith a handheld mass spectrometer.

FIG. 19 panels A-F show mass spectra of cocaine sprayed from sixdifferent types of paper (Whatman filter paper with different poresizes: (a) 3 μm, (b) 4-7 μm, (c) 8 μm, and (d) 11 μm, (e) glass fiberpaper and (f) chromatography paper). The spray voltage was 4.5 kV.

FIG. 20 panel (A) shows a schematic setup for characterizing the spatialdistribution of paper spray. Panel (B) is a 2D contour plot showing therelative intensity of m/z 304 when the probe is moved in the x-y planewith respect to the inlet of the mass spectrometer. Panel (C) is a graphshowing signal duration of m/z 304 when loading cocaine solution onpaper with different concentrations or volumes, or sealed by Teflonmembrane.

FIG. 21 is a set of MS spectra of pure chemical solutions and theircorresponding MS/MS spectra. Spectra were obtained for (A) serine, (B)methadone, (C) roxithromycin, and (D) bradykinin 2-9.

FIG. 22 is a set of mass spectra showing analysis of chemicals fromcomplex mixtures and direct analysis from surfaces without samplepreparation. Panels (A and B) are mass spectra of COCA-COLA (coladrink), which was directly analyzed on paper in both of (A) positive and(B) negative mode. Panel (C) is a mass spectrum of caffeine. Panel (D)is a mass spectrum of potassium benzoate. Panel (E) is a mass spectrumof acesulfame potassium. Panel (F) is a mass spectrum of caffeinedetected from urine. Panel (G) is a mass spectrum of heroin detecteddirectly from a desktop surface after swabbing of the surface by probesof then invention.

FIG. 23 panel (A) shows images of a probe of the invention used forblood analysis. In this embodiment, the porous material is paper. Thepanel on the left is prior to spotting with whole blood. The panel inthe middle is after spotting with whole blood and allowing the spot todry. The panel on the right is after methanol was added to the paper andallowed to travel through the paper. The panel on the right shows thatthe methanol interacts with the blood spot, causing analytes to travelto the tip of the paper for ionization and analysis. Panel (B) is a massspectrum of Atenolol from whole blood. Panel (C) is a mass spectrum ofheroin from whole blood.

FIG. 24 shows analysis of two dyes, methylene blue (m/z 284) and methylviolet (m/z 358.5), separated by TLC. Dye mixture solution (0.1 μl of a1 mg/mL solution) was applied onto the chromatography paper (4 cm×0.5cm) and dried before TLC and paper spray MS analysis.

FIG. 25 shows different shapes, thicknesses, and angles for probes ofthe invention. Panel (A) shows sharpness. Panel (B) shows angle of thetip. Panel (C) shows thickness of the paper. Panel (D) shows a devicewith multiple spray tips. Panel (E) shows a DBS card with micro spraytips fabricated with sharp needles.

FIG. 26 is a set of mass spectra of imatinib from human serum usingdirect spray from a C4 zip-tip of conical shape. Human serum samples(1.5 μL each) containing imatinib were passed through the porous C4extraction material three times and then 3 μL methanol was added ontothe zip-tip with 4 kV positive DC voltage applied to produce the spray.Panel (A) shows a MS spectrum for 5 μg/mL. Panel (B) shows a MS/MSspectrum for 5 ng/mL.

FIG. 27 panel (A) is a picture showing different tip angles for probesof the invention. From left to right, the angles are 30, 45, 90, 112,126 degree, respectively. Panel (B) is a graph showing the effect ofangle on MS signal intensity. All MS signals were normalized to the MSsignal using the 90 degree tip.

FIG. 28 panel (A) is a picture of a high-throughput probe device of theinvention. Panel (B) shows spray from a single tip of the device into aninlet of a mass spectrometer. Panel (C) is a set of mass spectra showingMS signal intensity in high-throughput mode.

FIG. 29 panel (A) is a schematic depicting a protocol for directanalysis of animal tissue using probes of the invention. Panels (Bthrough D) are mass spectra showing different chemicals detected in thetissue.

FIG. 30 panel (A) shows a mass spectral analysis of a dried serum spoton plain paper. Panel (B) shows a mass spectrum analysis of a driedserum sport on paper preloaded with betaine aldehyde (BA) chloride.Panel (C) shows a MS/MS analysis of reaction product [M+BA]⁺ (m/z488.6).

FIG. 31 shows MS/MS spectra recorded with modified (panel A) andunmodified (panel B) paper substrates.

FIG. 32 is a mass spectrum showing that ions can be generated using anegative ion source potential but positively charged ions aremass-analyzed.

FIG. 33 panel (A) is a schematic showing the design of a samplecartridge with volume control and overflowing vials. A soluble plug withinternal standard chemical is used to block the bottom of the volumecontrol vial. Panel (B) shows a step-by-step process of applying bloodsamples onto the cartridge to prepare a dried blood spot on paper from acontrolled volume of blood.

FIG. 34 panels (A and B) show mass spectra of agrochemicals that arepresent on a lemon peel purchased from a grocery store and swabbed withpaper.

FIG. 35 shows a design of a substrate for paper spray with multiplecorners. The angle of the corner to be used for spray is smaller thanthat of other corners.

FIG. 36 panels (A and B) show a spray tip fabricated on a piece ofchromatography paper using SU-8 2010 photoresist. Panel (C) shows a MSspectrum of methanol/water solution containing a mixture of asparagines.

DETAILED DESCRIPTION

A new method of generating ions from fluids and solids for massspectrometry analysis is described. Porous materials, such as paper(e.g. filter paper or chromatographic paper) or other similar materialsare used to hold and transfer liquids and solids, and ions are generateddirectly from the edges of the material when a high electric voltage isapplied to the material (FIG. 1). The porous material is kept discrete(i.e., separate or disconnected) from a flow of solvent, such as acontinuous flow of solvent. Instead, sample is either spotted onto theporous material or swabbed onto it from a surface including the sample.The spotted or swabbed sample is then connected to a high voltage sourceto produce ions of the sample which are subsequently mass analyzed. Thesample is transported through the porous material without the need of aseparate solvent flow. Pneumatic assistance is not required to transportthe analyte; rather, a voltage is simply applied to the porous materialthat is held in front of a mass spectrometer.

In certain embodiments, the porous material is any cellulose-basedmaterial. In other embodiments, the porous material is a non-metallicporous material, such as cotton, linen wool, synthetic textiles, orplant tissue. In still other embodiments, the porous material is paper.Advantages of paper include: cost (paper is inexpensive); it is fullycommercialized and its physical and chemical properties can be adjusted;it can filter particulates (cells and dusts) from liquid samples; it iseasily shaped (e.g., easy to cut, tear, or fold); liquids flow in itunder capillary action (e.g., without external pumping and/or a powersupply); and it is disposable.

In certain embodiments, the porous material is integrated with a solidtip having a macroscopic angle that is optimized for spray. In theseembodiments, the porous material is used for filtration,pre-concentration, and wicking of the solvent containing the analytesfor spray at the solid type.

In particular embodiments, the porous material is filter paper.Exemplary filter papers include cellulose filter paper, ashless filterpaper, nitrocellulose paper, glass microfiber filter paper, andpolyethylene paper. Filter paper having any pore size may be used.Exemplary pore sizes include Grade 1 (11 μm), Grade 2 (8 μm), Grade 595(4-7 μm), and Grade 6 (3 μm), Pore size will not only influence thetransport of liquid inside the spray materials, but could also affectthe formation of the Taylor cone at the tip. The optimum pore size willgenerate a stable Taylor cone and reduce liquid evaporation. The poresize of the filter paper is also an important parameter in filtration,i.e., the paper acts as an online pretreatment device. Commerciallyavailable ultra filtration membranes of regenerated cellulose, with poresizes in the low nm range, are designed to retain particles as small as1000 Da. Ultra filtration membranes can be commercially obtained withmolecular weight cutoffs ranging from 1000 Da to 100,000 Da.

Probes of the invention work well for the generation of micron scaledroplets simply based on using the high electric field generated at anedge of the porous material. In particular embodiments, the porousmaterial is shaped to have a macroscopically sharp point, such as apoint of a triangle, for ion generation. Probes of the invention mayhave different tip widths. In certain embodiments, the probe tip widthis at least about 5 μm or wider, at least about 10 μm or wider, at leastabout 50 μm or wider, at least about 150 μm or wider, at least about 250μm or wider, at least about 350 μm or wider, at least about 400μ orwider, at least about 450 μm or wider, etc. In particular embodiments,the tip width is at least 350 μm or wider. In other embodiments, theprobe tip width is about 400 μm. In other embodiments, probes of theinvention have a three dimensional shape, such as a conical shape.

As mentioned above, no pneumatic assistance is required to transport thedroplets. Ambient ionization of analytes is realized on the basis ofthese charged droplets, offering a simple and convenient approach formass analysis of solution-phase samples.

Sample solution is directly applied on the porous material held in frontof an inlet of a mass spectrometer without any pretreatment. Then theambient ionization is performed by applying a high potential on thewetted porous material. In certain embodiments, the porous material ispaper, which is a type of porous material that contains numerical poresand microchannels for liquid transport. The pores and microchannels alsoallow the paper to act as a filter device, which is beneficial foranalyzing physically dirty or contaminated samples.

In other embodiments, the porous material is treated to producemicrochannels in the porous material or to enhance the properties of thematerial for use as a probe of the invention. For example, paper mayundergo a patterned silanization process to produce microchannels orstructures on the paper. Such processes involve, for example, exposingthe surface of the paper totridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane to result insilanization of the paper. In other embodiments, a soft lithographyprocess is used to produce microchannels in the porous material or toenhance the properties of the material for use as a probe of theinvention. In other embodiments, hydrophobic trapping regions arecreated in the paper to pre-concentrate less hydrophilic compounds.

Hydrophobic regions may be patterned onto paper by usingphotolithography, printing methods or plasma treatment to definehydrophilic channels with lateral features of 200˜1000 μm. See Martinezet al. (Angew. Chem. Int. Ed. 2007, 46, 1318-1320); Martinez et al.(Proc. Natl Acad. Sci. USA 2008, 105, 19606-19611); Abe et al. (Anal.Chem. 2008, 80, 6928-6934); Bruzewicz et al. (Anal. Chem. 2008, 80,3387-3392); Martinez et al. (Lab Chip 2008, 8, 2146-2150); and Li et al.(Anal. Chem. 2008, 80, 9131-9134), the content of each of which isincorporated by reference herein in its entirety. Liquid samples loadedonto such a paper-based device can travel along the hydrophilic channelsdriven by capillary action.

Another application of the modified surface is to separate orconcentrate compounds according to their different affinities with thesurface and with the solution. Some compounds are preferably absorbed onthe surface while other chemicals in the matrix prefer to stay withinthe aqueous phase. Through washing, sample matrix can be removed whilecompounds of interest remain on the surface. The compounds of interestcan be removed from the surface at a later point in time by otherhigh-affinity solvents. Repeating the process helps desalt and alsoconcentrate the original sample.

Methods and systems of the invention use a porous material, e.g., paper,to hold and transport analytes for mass spectral analysis. Analytes insamples are pre-concentrated, enriched and purified in the porousmaterial in an integrated fashion for generation of ions withapplication of a high voltage to the porous material. In certainembodiments, a discrete amount of transport solution (e.g., a droplet ora few droplets) is applied to assist movement of the analytes throughthe porous material. In certain embodiments, the analyte is already in asolution that is applied to the porous material. In such embodiments, noadditional solvent need be added to the porous material. In otherembodiments, the analyte is in a powdered sample that can be easilycollected by swabbing a surface. Systems and methods of the inventionallow for analysis of plant or animal tissues, or tissues in livingorganisms.

Methods and systems of the invention can be used for analysis of a widevariety of small molecules, including epinephrine, serine, atrazine,methadone, roxithromycin, cocaine and angiotensin I. All display highquality mass and MS/MS product ion spectra (see Examples below) from avariety of porous surfaces. Methods and systems of the invention allowfor use of small volumes of solution, typically a few μL, with analyteconcentrations on the order of 0.1 to 10 μg/mL (total amount analyte 50pg to 5 ng) and give signals that last from one to several minutes.

Methods and systems of the invention can be used also for analysis of awide variety of biomolecules, including proteins and peptides. Methodsof the invention can also be used to analyze oligonucleotides from gels.After electrophoretic separation of oligonucleotides in the gel, theband or bands of interest are blotted with porous material using methodsknown in the art. The blotting results in transfer of at least some ofthe oligonucleotides in the band in the gel to the porous material. Theporous material is then connected to a high voltage source and theoligonucleotides are ionized and sprayed into a mass spectrometer formass spectral analysis.

Methods and systems of the invention can be used for analysis of complexmixtures, such as whole blood or urine. The typical procedure for theanalysis of pharmaceuticals or other compounds in blood is a multistepprocess designed to remove as many interferences as possible prior toanalysis. First, the blood cells are separated from the liquid portionof blood via centrifugation at approximately 1000×g for 15 minutes(Mustard, J. F.; Kinlough-Rathbone, R. L.; Packham, M. A. Methods inEnzymology; Academic Press, 1989). Next, the internal standard is spikedinto the resulting plasma and a liquid-liquid or solid-phase extractionis performed with the purpose of removing as many matrix chemicals aspossible while recovering nearly all of the analyte (Buhrman, D. L.;Price, P. I.; Rudewicz, P. J. Journal of the American Society for MassSpectrometry 1996, 7, 1099-1105). The extracted phase is typically driedby evaporating the solvent and then resuspended in the a solvent used asthe high performance liquid chromatography (HPLC) mobile phase(Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M., Ithaca, N.Y.,Jul. 23-25 1997; 882-889). Finally, the sample is separated in thecourse of an HPLC run for approximately 5-10 minutes, and the eluent isanalyzed by electrospray ionization-tandem mass spectrometry(Hopfgartner, G.; Bourgogne, E. Mass Spectrometry Reviews 2003, 22,195-214).

Methods and systems of the invention avoid the above sample work-upsteps. Methods and systems of the invention analyze a dried blood spotsin a similar fashion, with a slight modification to the extractionprocedure. First, a specialized device is used to punch out identicallysized discs from each dried blood spot. The material on these discs isthen extracted in an organic solvent containing the internal standard(Chace, D. H.; Kalas, T. A.; Naylor, E. W. Clinical Chemistry 2003, 49,1797-1817). The extracted sample is dried on the paper substrate, andthe analysis proceeds as described herein.

Examples below show that methods and systems of the invention candirectly detect individual components of complex mixtures, such ascaffeine in urine, 50 pg of cocaine on a human finger, 100 pg of heroinon a desktop surface, and hormones and phospholipids in intact adrenaltissue, without the need for sample preparation prior to analysis (SeeExamples below). Methods and systems of the invention allow for simpleimaging experiments to be performed by examining, in rapid succession,needle biopsy tissue sections transferred directly to paper.

Analytes from a solution are applied to the porous material forexamination and the solvent component of the solution can serve as theelectrospray solvent. In certain embodiments, analytes (e.g., solid orsolution) are pre-spotted onto the porous material, e.g., paper, and asolvent is applied to the material to dissolve and transport the analyteinto a spray for mass spectral analysis.

In certain embodiments, a solvent is applied to the porous material toassist in separation/extraction and ionization. Any solvents may be usedthat are compatible with mass spectrometry analysis. In particularembodiments, favorable solvents will be those that are also used forelectrospray ionization. Exemplary solvents include combinations ofwater, methanol, acetonitrile, and THF. The organic content (proportionof methanol, acetonitrile, etc. to water), the pH, and volatile salt(e.g. ammonium acetate) may be varied depending on the sample to beanalyzed. For example, basic molecules like the drug imatinib areextracted and ionized more efficiently at a lower pH. Molecules withoutan ionizable group but with a number of carbonyl groups, like sirolimus,ionize better with an ammonium salt in the solvent due to adductformation.

In certain embodiments, a multi-dimensional approach is undertaken. Forexample, the sample is separated along one dimension, followed byionization in another dimension. In these embodiments, separation andionization can be individually optimized, and different solvents can beused for each phase.

In other embodiments, transporting the analytes on the paper isaccomplished by a solvent in combination with an electric field. When ahigh electric potential is applied, the direction of the movement of theanalytes on paper is found to be related to the polarity of theircharged forms in solution. Pre-concentration of the analyte before thespray can also be achieved on paper by placing an electrode at a pointon the wetted paper. By placing a ground electrode near the paper tip, astrong electric field is produced through the wetted porous materialwhen a DC voltage is applied, and charged analytes are driven forwardunder this electric field. Particular analytes may also be concentratedat certain parts of the paper before the spray is initiated.

In certain embodiments, chemicals are applied to the porous material tomodify the chemical properties of the porous material. For example,chemicals can be applied that allow differential retention of samplecomponents with different chemical properties. Additionally, chemicalscan be applied that minimize salt and matrix effects. In otherembodiments, acidic or basic compounds are added to the porous materialto adjust the pH of the sample upon spotting. Adjusting the pH may beparticularly useful for improved analysis of biological fluids, such asblood. Additionally, chemicals can be applied that allow for on-linechemical derivatization of selected analytes, for example to convert anon-polar compound to a salt for efficient electrospray ionization.

In certain embodiments, the chemical applied to modify the porousmaterial is an internal standard. The internal standard can beincorporated into the material and released at known rates duringsolvent flow in order to provide an internal standard for quantitativeanalysis. In other embodiments, the porous material is modified with achemical that allows for pre-separation and pre-concentration ofanalytes of interest prior to mass spectrum analysis.

The spray droplets can be visualized under strong illumination in thepositive ion mode and are comparable in size to the droplets emittedfrom a nano-electrospray ion sources (nESI). In the negative ion mode,electrons are emitted and can be captured using vapor phase electroncapture agents like benzoquinone. Without being limited by anyparticular theory or mechanism of action, it is believed that the highelectric field at a tip of the porous material, not the fields in theindividual fluid channels, is responsible for ionization.

The methodology described here has desirable features for clinicalapplications, including neotal screening, therapeutic drug monitoringand tissue biopsy analysis. The procedures are simple and rapid. Theporous material serves a secondary role as a filter, e.g., retainingblood cells during analysis of whole blood. Significantly, samples canbe stored on the porous material and then analyzed directly from thestored porous material at a later date without the need transfer fromthe porous material before analysis. Systems of the invention allow forlaboratory experiments to be performed in an open laboratoryenvironment.

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES

The following examples are intended to further illustrate certainembodiments of the invention, and are not to be construed to limit thescope of the invention. Examples herein show that mass spectrometryprobes of the invention can ionize chemical and biological samples,allowing for subsequent mass analysis and detection. An exemplary probewas constructed as a paper triangle, which was used to generate micronscale droplets by applying a high potential on the paper. The analyteswere ionized from these electrically charged droplets and transportedinto a conventional mass spectrometer.

Examples below show that a wide range of samples could be directlyanalyzed in the ambient environment by probes of the invention in bothof pure state and complex mixtures. The results showed that paper-basedspray has the following benefits: it operated without sheath gas, i.e. ,few accessories were required for in situ analysis; biological samples(dried blood, urine) could be stored on the precut filter papers formonths before analysis; filter paper minimized matrix effects seen withelectrospray or nano electrospray in many samples (blood cells, salt andproteins) and enhanced the MS signal of chemicals in complex samples;powdered samples were easily collected by swabbing surfaces using paperpieces and then directly analyzed; the paper could be pretreated tocontain internal standards that were released at known rates duringsolvent flow in quantitative analysis; and the paper could be pretreatedto contain matrix suppression or absorption sites or to perform ionexchange or to allow on-line chemical derivatization of selectedanalytes.

Detection of most analytes was achieved as low as ppb levels (whenexamined as solutions) or in the low ng to pg range (when solids wereexamined) and the detection time was less than one minute. CertainExamples below provide a protocol for analyzing a dried blood spot,which can also be used for in situ analysis of whole blood samples. Thedried blood spot method is also demonstrated to be compatible with thestorage and transport of blood sample for blood screening and otherclinical tests.

Devices of the invention integrated the capabilities of sampling,pre-separation, pre-concentration and ionization. Methods and systems ofthe invention simplify the problem of sample introduction in massanalyzers.

Example 1 Construction of an MS Probe

Filter paper was cut into triangular pieces with dimensions of 10 mmlong and 5 mm wide and used as a sprayer (FIG. 1). A copper clip wasattached to the paper, and the paper was oriented to face an inlet of amass spectrometer (FIG. 1). The copper clip was mounted on a 3D movingstage to accurately adjust its position. A high voltage was applied tothe copper clip and controlled by a mass spectrometer to generateanalyte ions for mass detection.

Samples were directly applied to the paper surface that served as asample purification and pre-concentration device. Filter paper allowedliquid samples to move through the hydrophilic network driven bycapillary action and electric effects and to transport them to the tipof the paper. Separation could take place during this transport process.Sample solution was sprayed from the tip and resulted in ionization andMS detection when a high voltage (˜4.5 kV) was applied to the papersurface.

All experiments were carried out with a Finnigan LTQ mass spectrometer(Thermo Electron, San Jose, Calif.). The typical temperature of thecapillary inlet was set at 150° C. while 30° C. for heroin detection.The lens voltage was set at 65 V for sample analysis and 240 V forsurvival yield experiment. Tandem mass spectra were collected usingcollision-induced dissociation (CID) to identify analytes in testedsamples, especially for complex mixtures and blood samples.

Example 2 Spray Generation

Spray was produced by applying a high potential on the wetted papertriangle. One paper triangle was placed in front of the inlet of LTQwith its sharp tip facing to the inlet, separated by 3 mm or more.Typically, 10 uL sample solution was applied to wet the paper triangle.The solution can wet or saturate the paper or form a thin layer ofliquid film on the surface of the paper. A high potential (3-5 kV) wasapplied between the paper triangle and mass inlet to generate anelectric field, which induced a charge accumulation on the liquid at thetip of paper triangle. The increasing coulombic force breaks the liquidto form charged droplets and then the solvent evaporated during theflight of droplets from the paper tip to the mass analyzer. Paper sprayrequired no sheath gas, heating or any other assistance to remove thesolvent.

When liquid accumulated on the paper triangle, a Taylor cone wasobserved at the tip when examined with a microscope. The droplets formedwere clearly visible under strong illumination. The Taylor cone andvisible spray disappeared after a short time of evaporation and spray.However, the mass signal lasted for a much longer period (severalminutes). This revealed that the paper triangle could work in two modesfor mass analysis. In a first mode, the liquid was transported insidethe paper at a rate faster than the liquid could be consumed as spray atthe paper tip, resulting in a large cone being formed at the paper tipand droplets being generated. In a second mode, the liquid transportinside the paper was not able to move at a rate fast enough to keep upwith the spray consumption, and droplets were not visible. However, itwas observed that ionization of analytes did take place. The first modeprovided ESI like mass spectra and the second mode provided spectra withsome of the features APCI spectra. In the latter case, the papertriangle played a role analogous to a conductive needle to generate ahigh electric field to ionize the molecules in the atmosphere. It wasobserved that the mass signal in the first mode was stronger than themass signal in the second mode by approximately two orders of magnitudeunder the conditions and for the samples tested.

Example 3 Probe Considerations

Probe Materials

A number of porous materials were tested to generate charged dropletsfor mass spectrometry. The materials were shaped into triangles havingsharp tips and sample solution was then applied to the constructedprobes. Data herein show that any hydrophilic and porous substrate couldbe used successfully, including cotton swab, textile, plant tissues aswell as different papers. The porous network or microchannels of thesematerials offered enough space to hold liquid and the hydrophilicenvironment made it possible for liquid transport by capillary action.Hydrophobic and porous substrates could also be used successfully withproperly selected hydrophobic solvents.

For further investigation, six kinds of commercialized papers wereselected and qualitatively tested to evaluate their capabilities inanalyte detection. Filter papers and chromatography paper were made fromcellulose, while glass microfiber filter paper was made from glassmicrofiber. FIG. 19 shows the mass spectra of cocaine detection on thosepapers. The spectrum of glass fiber paper (FIG. 19 panel E) was uniquebecause the intensity of background was two orders of magnitude lowerthan other papers and the cocaine peak (m/z, 304) could not beidentified.

It was hypothesized that the glass fiber paper was working on mode IIand prohibiting efficient droplet generation, due to the relative largethickness (˜2 mm). This hypothesis was proved by using a thin layerpeeled from glass fiber paper for cocaine detection. In that case, theintensity of the background increased and a cocaine peak was observed.All filter papers worked well for cocaine detection, (FIG. 19 panelsA-D). Chromatography paper showed the cleanest spectrum and relativehigh intensity of cocaine (FIG. 19 panel F).

Probe Shape and Tip Angle

Many different probe shapes were investigated with respect to generatingdroplets. A preferred shape of the porous material included at least onetip. It was observed that the tip allowed ready formation of a Taylorcone. A probe shape of a triangle was used most often. As shown in FIG.25 panels (A-C), the sharpness of the tip, the angle of the tip (FIG. 27panels A and B), and the thickness of the paper substrate could effectthe spray characteristics. The device of a tube shape with multiple tips(FIG. 25 panel D) is expected to act as a multiple-tip sprayer, whichshould have improved spray efficiency. An array of micro sprayers canalso be fabricated on a DBS card using sharp needles to puncture thesurface (FIG. 25 panel E).

Example 4 Configuration of Probe with Inlet of a Mass Spectrometer

A paper triangle was mounted on a 2D moving stage to determine how themass signal was affected by the relative positions of the paper triangleand the mass spectrometer inlet. The paper triangle was moved 8 cm inthe y-direction in a continuous manner and 3 cm in the x-direction witha 2 mm increment for each step (FIG. 20 panel A). Cocaine solution (1ug/mL, methanol/water, 1:1 v/v) was continuously fed onto the papersurface. The mass spectrum was continuously recorded during the entirescan. A contour plot of the peak intensity of protonated cocaine (m/z,304) was created from the normalized data extracted from the massspectrum (FIG. 20 panel B). The contour plot shows that it was notnecessary for the paper triangle to be placed directly in-line with theinlet of the mass spectrometer to generate droplets.

Spray duration was also tested (FIG. 20 Panel C). Paper triangles (size10 mm, 5 mm) were prepared. First, 10 uL solutions were applied on thepaper triangles with different concentration of 0.1, 1 and 10 ug/mL. Thespray time for each paper was just slightly varied by the difference ofconcentration. After that, 1 ug/mL cocaine solutions were applied on thepaper triangles with different volumes of 5 uL, 10 uL and 15 uL. Thespray times showed a linear response followed by the increasing samplevolumes.

In another test, the paper was sealed with a PTFE membrane to preventevaporation of solution, which prolonged the spray time by about threetimes. These results indicate that paper spray offers long enough timeof spray for data acquisition even using 5 uL solution, and theintensity of signal is stable during the entire spray period.

Example 5 Separation and Detection

Probes of the invention include a porous material, such as paper, thatcan function to both separate chemicals in biological fluids before insitu ionization by mass spectrometry. In this Example, the porousmaterial for the probe was chromatography paper. As shown in FIG. 24, amixture of two dyes was applied to the paper as a single spot. The dyeswere first separated on the paper by TLC (thin layer chromatograph) andthe separated dyes were examined using MS analysis by methods of theinvention with the paper pieces cut from the paper media (FIG. 24). Datashow the separate dyes were detected by MS analysis (FIG. 24).

The chromatography paper thus allowed for sample collection, analyteseparation and analyte ionization. This represents a significantsimplification of coupling chromatography with MS analysis.Chromatography paper is a good material for probes of the inventionbecause such material has the advantage that solvent movement is drivenby capillary action and there is no need for a syringe pump. Anotheradvantage is that clogging, a serious problem for conventionalnanoelectrospray sources, is unlikely due to its multi-porouscharacteristics. Therefore, chromatography paper, a multi-porousmaterial, can be used as a microporous electrospray ionization source.

Example 6 Pure Compounds: Organic Drugs, Amino Acids, and Peptides

As already described, probes and methods of the invention offer a simpleand convenient ionization method for mass spectrometry. Paper triangleswere spotted with different compounds and connected to a high voltagesource to produce ions. All experiments were carried out with a FinniganLTQ mass spectrometer (Thermo Electron, San Jose, Calif.). Data hereinshow that a variety of chemicals could be ionized in solution phase,including amino acid, therapeutic drugs, illegal drugs and peptides.

FIG. 2 panel (A) shows an MS spectrum of heroin (concentration: 1 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 2 panel (B) shows MS/MS spectrum of heroin(concentration: 1 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 3 panel (A) shows MS spectrum of caffeine (concentration: 10 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 3 panel (B) shows MS/MS spectrum of caffeine(concentration: 10 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)). Peak 167 also exists in the blank spectrum with solvent andwithout caffeine.

FIG. 4 panel (A) shows MS spectrum of benzoylecgonine (concentration: 10ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) usingprobes of the invention. FIG. 4 panel (B) shows MS/MS spectrum ofbenzoylecgonine (concentration: 10 ppb, volume: 10 μl, solvent:MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 5 panel (A) shows MS spectrum of serine (concentration: 1 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 5 panel (B) shows MS/MS spectrum of serine(concentration: 100 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)). Peak 74 and 83 also exist in the blank spectrum with solventand without serine. FIG. 21 panel (A) shows MS spectrum of serine (m/z,106) using probes of the invention. Panel (A) also shows MS/MS spectrumof serine (m/z, 106).

FIG. 21 Panel (B) shows MS spectrum of methadone (m/z, 310) using probesof the invention. Panel (B) also shows MS/MS spectrum of methadone (m/z,310). Panel (C) shows MS spectrum of roxithromycin (m/z, 837) usingprobes of the invention. Panel (B) also shows MS/MS spectrum ofroxithromycin (m/z, 837).

FIG. 6 panel (A) shows MS spectrum of peptide bradykinin2-9(concentration: 10 ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)) using probes of the invention. FIG. 6 panel (B) shows MS/MSspectrum of bradykinin2-9 (concentration: 1 ppm, volume: 10 μl, solvent:MeOH/H₂O/HOAc (50:49:1, v/v/v)). The hump in the spectrum is assumed tobe caused by polymers, such as polyethylene glycol (PEG), which arefrequently added to materials in industry. FIG. 21 panel (D) shows MSspectrum of bradykinin 2-9 (m/z, 453) using probes of the invention.Panel (D) also shows MS/MS spectrum of bradykinin 2-9 (m/z, 453). PanelD further shows adduct ions [M+H] (m/z, 904), [M+2H]²⁺ (m/z, 453),[M+H+Na]²⁺ (m/z, 464) and [M+2Na]²⁺ (m/z, 475). The m/z 453 peak wasdouble charged adduct ion confirmed by the MS/MS spectrum.

FIG. 11 is an MS spectra showing the difference between peptide analysis(10 ppm of bradykinin 2-9) on (A) paper slice and (B) PVDF membraneusing the same parameters (˜2 kV, Solvent: MeOH:H₂O=1:1).

Data herein show that probes of the invention work well over themass/charge range from 50 to over 1000 for detection of pure compounds.Data further shows that detection was achieved down to as low as 1 ng/mLfor most chemicals, including illegal drugs, such as heroin, cocaine andmethadone.

Example 7 Complex Mixtures

Complex mixtures such as urine, blood, and cola drink were examinedusing methods, devices, and systems of the invention. All experimentswere carried out with a Finnigan LTQ mass spectrometer (Thermo Electron,San Jose, Calif.).

FIG. 7 panel (A) shows an MS/MS spectrum that shows that heroin wasdetected from whole blood sample by a “spot” method. 0.4 μl of wholeblood sample containing 200 ppb heroin was applied on the center of thetriangle paper to form a 1 mm² blood spot. After the spot was dry, 10 μlof solvent (MeOH/H₂O/HOAc (50:49:1, v/v/v)) was applied to the rear endof the triangle paper. Due to the capillary effect, the solvent movedforward and dissolved the chemicals in the blood spot. Finally,electrospray occurred when the solvent reached the tip of the paper. Todemonstrate the effectiveness of the “blood spot” method mentionedabove, the whole blood was added on the paper for electrospray directly.MS/MS spectrum showed that heroin was not detected from 10 μl of wholeblood sample, even when the concentration was as high as 20 ppm (FIG. 7panel B).

FIG. 8 panel (A) shows an MS/MS spectrum that shows that heroin can bedetected from raw urine sample by a “spot” method. 0.4 μl of raw urinesample containing 100 ppb heroin was applied on the center of thetriangle paper to form a 1 mm² urine spot. After the spot was dry, 10 μlof solvent (MeOH/H₂O/HOAc (50:49:1, v/v/v)) was applied to the rear endof the triangle paper. Due to the capillary effect, the solvent movedforward and dissolved the chemicals in the blood spot. Finally,electrospray occurred when the solvent reached the tip of the paper. Todemonstrate the effectiveness of the “spot” method mentioned above, theraw urine was added on the paper for electrospray directly. MS/MSspectrum showed heroin was not detected from 10 μl of raw urine samplewhen concentration was 100 ppb (FIG. 8 panel B).

FIG. 9 panel (A) is an MS spectrum showing that caffeine was detectedfrom a cola drink without sample preparation. FIG. 9 panel (B) is an MSspectrum showing that caffeine was detected from coffee powder. A papertriangle was used to collect the coffee powder from a coffee bag byswabbing the surface.

FIG. 22 panels (A-B) show the spectra of COCA-COLA (cola drink),analyzed in positive mode and negative mode, respectively. The peak ofprotonated caffeine, m/z 195, identified in MS/MS spectrum, wasdominated in the mass spectrum in positive mode due to the highconcentration of caffeine (100 ug/mL) in this drink (Panel C). Two highconcentrated compounds, potassium benzoate and acesulfame potassium wereidentified in the MS/MS spectrum in negative mode (Panels D-E).

FIG. 22 panel F shows spectra of caffeine in urine from a person who haddrunk COCA-COLA (cola drink) two hours before the urine collection.Urine typically contains urea in very high concentration, which is alsoeasily ionized. Therefore, protonated urea [m/z, 61] and urea dimmer[m/z, 121] dominated the MS spectrum. However, the protonated caffeinewas identified in the MS/MS spectrum, which showed good signal to noiseratio in the urine sample.

FIG. 10 shows MS spectra of urine taken for analysis without samplepreparation. FIG. 10 panel (A) is a mass spectra of caffeine that wasdetected in urine from a person who had consumed coffee. FIG. 10 panel(B) is a mass spectra showing that caffeine was not detected in urinefrom a person who had not consumed any coffee.

FIG. 22 panel G shows the MS spectrum of heroin (m/z, 370) collected asa swabbed sample. A 5 uL solution containing 50 ng heroin was spotted ona 1 cm² area of a desktop. The paper triangle was wetted and used toswab the surface of the desktop. The paper triangle was then connectedto the high voltage source for mass detection. This data shows thatprobes of the invention can have dual roles of ionization source as wellas a sampling device for mass detection. Trace sample on solid surfacecould be simply collected by swabbing the surface using probes of theinvention. Dust and other interferences were also collected on the papertriangle, but the heroin could be directly detected from this complexmatrix.

Example 8 Plant Tissue Direct Analysis by ESI Without Extraction

FIG. 12 shows direct MS spectra of plant tissues using sliced tissues offour kinds of plants. (A) Onion, (B) Spring onion, and two differentleaves (C) and (D).

FIG. 13 shows an MS/MS spectra of Vitamin C analysis (A) direct analysisof onion without sample preparation, (B) using standard solution.

Example 9 Whole Blood and Other Biofluids

Body fluids, such as plasma, lymph, tears, saliva, and urine, arecomplex mixtures containing molecules with a wide range of molecularweights, polarities, chemical properties, and concentrations. Monitoringparticular chemical components of body fluids is important in a numberof different areas, including clinical diagnosis, drug development,forensic toxicology, drugs of abuse detection, and therapeutic drugmonitoring. Tests of blood, including the derived fluids plasma andserum, as well as on urine are particularly important in clinicalmonitoring.

A wide variety of chemicals from blood are routinely monitored in aclinical setting. Common examples include a basic metabolic panelmeasuring electrolytes like sodium and potassium along with urea,glucose, and creatine and a lipid panel for identifying individuals atrisk for cardiovascular disease that includes measurements of totalcholesterol, high density lipoprotein (HDL), low density lipoprotein(LDL), and triglycerides. Most laboratory tests for chemicals in bloodare actually carried out on serum, which is the liquid component ofblood separated from blood cells using centrifugation. This step isnecessary because many medical diagnostic tests rely on colorimetricassays and therefore require optically clear fluids. Aftercentrifugation, detection of the molecule of interest is carried in anumber of ways, most commonly by an immunoassay, such as anenzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), oran enzyme assay in which the oxidation of the molecule of interest by aselective enzyme is coupled to a reaction with a color change, such asthe tests for cholesterol (oxidation by cholesterol oxidase) or glucose(oxidation by glucose oxidase).

There is considerable interest in the pharmaceutical sciences in thestorage and transportation of samples of whole blood as dried bloodspots on paper (N. Spooner et al. Anal Chem., 2009, 81, 1557). Mosttests for chemicals found in blood are carried out on a liquid sample,typically serum or plasma isolated from the liquid whole blood. Therequired storage, transportation, and handling of liquid blood or bloodcomponents present some challenges. While blood in liquid form isessential for some tests, others can be performed on blood or other bodyfluids that have been spotted onto a surface (typically paper) andallowed to dry.

Probes and methods of the invention can analyze whole blood without theneed for any sample preparation. The sample was prepared as follows. 0.4uL blood was directly applied on the center of paper triangle and leftto dry for about 1 min. to form a dried blood spot (FIG. 23 panel A). 10uL methanol/water (1:1, v/v) was applied near the rear end of the papertriangle. Driven by capillary action, the solution traveled across thepaper wetting it throughout its depth. As the solution interacted withthe dried blood spot, the analytes from the blood entered the solutionand were transported to the tip of the probe for ionization (FIG. 23panel A). The process of blood sample analysis was accomplished in about2 min.

Different drugs were spiked into whole blood and the blood was appliedto probes of the invention as described above. Detection of differentdrugs is described below.

Imatinib (GLEEVEC), a 2-phenylaminopyrimidine derivative, approved bythe FDA for treatment of chronic myelogenous leukemia, is efficaciousover a rather narrow range of concentrations. Whole human blood, spikedwith imatinib at concentrations including the therapeutic range, wasdeposited on a small paper triangle for analysis (FIG. 14, panel A). Thetandem mass spectrum (MS/MS, FIG. 14 panel B) of protonated imatinib,m/z 494, showed a single characteristic fragment ion. Quantitation ofimatinib in whole blood was achieved using this signal and that for aknown concentration of imatinib-d8 added as internal standard. Therelative response was linear across a wide range of concentrations,including the entire therapeutic range (FIG. 14 panel C).

Atenolol, a β-blocker drug used in cardiovascular diseases, was testedusing the dried blood spot method to evaluate paper spray for wholeblood analysis. Atenolol was directly spiked into whole blood at desiredconcentrations and the blood sample was used as described above forpaper spray. The protonated atenolol of 400 pg (1 ug/mL atenolol in 0.4uL whole blood) in dried blood spot was shown in mass spectra, and theMS/MS spectra indicated that even 20 pg of atenolol (50 ug/mL atenololin 0.4 uL whole blood) could be identified in the dried blood spot (FIG.23 panel B).

FIG. 23 panel (C) is a mass spectra of heroin in whole blood. Dataherein show that 200 pg heroin in dried blood spot could be detectedusing tandem mass.

It was also observed that the paper medium served a secondary role as afilter, retaining blood cells. Significantly, samples were analyzeddirectly on the storage medium rather than requiring transfer from thepaper before analysis. All experiments were done in the open labenvironment. Two additional features indicated that the methodology hadthe potential to contribute to increasing the use of mass spectrometryin primary care facilities: blood samples for analysis were drawn bymeans of a pinprick rather than a canula; and the experiment was readilyperformed using a handheld mass spectrometer (FIG. 18 and Example 10below).

Example 10 Handheld Mass Spectrometer

Systems and methods of the invention were compatible with a handheldmass spectrometer. Paper spray was performed using a handheld massspectrometer (Mini 10, custom made at Purdue University). Analysis ofwhole blood spiked with 10 μg/mL atenolol. Methanol/water (1:1; 10 μL)was applied to the paper after the blood (0.4 uL) had dried (˜1 min) togenerate spray for mass detection (FIG. 18). The inset shows thatatenolol could readily be identified in whole blood using tandem massspectrum even when the atenolol amount is as low as 4 ng.

Example 11 Angiotensin I

FIG. 15 is a paper spray mass spectrum of angiotensin I solution(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO: 1), 10 μL, 8 μg/mLin methanol/water, 1:1, v/v) on chromatography paper (spray voltage, 4.5kV). The inset shows an expanded view over the mass range 630-700. Theprotonated ([M+2H]²⁺) and sodium-adduct ions ([M+H+Na]²⁺, [M+2Na]²⁺) arethe major ionic species.

Example 12 Agrochemicals on Fruit

Sample collection by paper wiping followed by analysis using probes ofthe invention was used for fast analysis of agrochemicals on fruit.Chromatography paper (3×3 cm) wetted with methanol was used to wipe a 10cm² area on the peel of a lemon purchased from a grocery store. Afterthe methanol had dried, a triangle was cut from the center of the paperand used for paper spray by applying 10 μL methanol/water solution. Thespectra recorded (FIG. 34 panels A-B) show that a fungicide originallyon the lemon peel, thiabendazole (m/z 202 for protonated molecular ionand m/z 224 for sodium adduct ion), had been collected onto the paperand could be identified easily with MS and confirmed using MS/MSanalysis. Another fungicide imazalil (m/z 297) was also observed to bepresent.

Example 13 Tumor Sample

Systems and methods of the invention were used to analyze human prostatetumor tissue and normal tissue. Tumor and adjacent normal tissuesections were 15 μm thick and fixed onto a glass slide for an imagingstudy using desorption electrospray ionization (DESI). A metal needlewas used to remove a 1 mm²×15 μm volume of tissue from the glass slidefrom the tumor region and then from the normal region and place themonto the surface of the paper triangle for paper spray analysis.

A droplet of methanol/water (1:1 v:v; 10 μl) was added to the paper assolvent and then 4.5 kV positive DC voltage applied to produce thespray. Phospholipids such as phosphatidylcholine (PC) and sphingomyelin(SM) were identified in the spectrum (FIG. 17 panels A and B). The peakof [PC(34:1)+K]⁺ at m/z 798 was significantly higher in tumor tissue andpeaks [SM(34:1)+Na]⁺ at m/z 725, [SM(36:0)+Na]⁺ at m/z 756, and[SM(36:4)+Na]⁺ at m/z 804 were significantly lower compared with normaltissue.

Example 14 Therapeutic Drug Monitoring

The administration of a drug depends on managing the appropriate dosingguidelines for achievement of a safe and effective outcome. Thisguideline is established during clinical trials where thepharmacokinetics (PK) and pharmacodynamics (PD) of the drug are studied.Clinical trials use PK-PD studies to establish a standard dose, whichmay be fixed or adjusted according formulas using variables like bodymass, body surface area, etc. However, the drug exposure, i.e. theamount of drug circulating over time, is influenced by a number offactors that vary from patient to patient. For example, an individuals'metabolic rate, the type and level of plasma proteins, and pre-existingconditions such as renal and/or hepatic impairment all play a role inaffecting the exposure of the drug in vivo. Further, administration of adrug in combination with other medications may also affect exposure. Asa result, it is often difficult to predict and prescribe an optimumregimen of drug administration.

Over- or underexposure to a drug can lead to toxic effects or decreasedefficacy, respectively. To address these concerns, therapeutic drugmonitoring (TDM) can be employed. TDM is the measurement of active druglevels in the body followed by adjustment of drug dosing or schedules toincrease efficacy and/or decrease toxicity. TDM is indicated when thevariability in the pharmacokinetics of a drug is large relative to thetherapeutic window, and there is an established relationship betweendrug exposure and efficacy and/or toxicity. Another requirement for TDMis that a sufficiently precise and accurate assay for the activeingredient must be available. Immunoassays and liquid chromatographymass spectrometry (LC-MS) are commonly used methods for TDM. Incomparison with immunoassay, LC-MS has advantages which include wideapplicability, high sensitivity, good quantitation, high specificity andhigh throughput. Probes of the invention may be coupled with standardmass spectrometers for providing point-of-care therapeutic drugmonitoring.

The drug Imatinib (GLEEVEC in USA and GLIVEC in Europe/Australia, forthe treatment of chronic myelogenous leu-kemia) in a dried blood spotwas analyzed using paper spray and a lab-scale LTQ mass spectrometer.Quantitation of Imatinib in whole blood was achieved using the MS/MSspectra with a known concentration of Imatinib-d8 being used as theinternal standard (FIG. 14 panel C). The relative response was linearacross a wide range of concentrations, including the entire therapeuticrange (FIG. 14 panel C).

Example 15 High-Throughout Detection

Multiple-tip devices were fabricated and applied for high throughputanalysis (FIG. 28 panel A). The multiple-tip device was a set of papertriangles all connected to a single copper strip (FIG. 28 panel A). Anelectrode was connected to the copper strip. Multiple samples were puton a single paper substrate and analyzed in series using themultiple-tip probe (FIG. 28 panels B-C). Each tip was pre-loaded with0.2 uL methanol/water containing 100 ppm sample (cocaine or caffeine)and dried. Then the whole multiple-tip device was moved on a movingstage from left to right with constant velocity and 7 uL methanol/waterwas applied from the back part for each tip during movement.

To prevent the contaminant during spray, blanks were inserted betweentwo sample tips. FIG. 28 panel (C) shows the signal intensity for thewhole scanning. From total intensity, six tips gave six individual highsignal peaks. For cocaine, peaks only appeared when tip 2 and tip 6 werescanned. For caffeine, the highest peak came from tip 4, which wasconsistent with the sample loading sequence.

Example 16 Tissue Analysis

Direct analysis of chemicals in animal tissue using probes of theinvention was performed as shown in FIG. 29 panel (A). A small sectionsof tissue were removed and placed on a paper triangle. Methanol/water(1:1 v:v; 10 μl) was added to the paper as solvent and then 4.5 kVpositive DC voltage was applied to produce the spray for MS analysis.Protonated hormone ions were observed for porcine adrenal gland tissue(1 mm³, Panel (B)). FIG. 16 is a mass spectrum showing direct analysisof hormones in animal tissue by paper spray. A small piece of pigadrenal gland tissue (1 mm×1 mm×1 mm) was placed onto the paper surface,MeOH/water (1:1 v:v; 10 μl) was added and a voltage applied to the paperto produce a spray. The hormones epinephrine and norepinephrine wereidentified in the spectrum; at high mass the spectrum was dominated byphospolipid signals.

Lipid profiles were obtained for human prostate tissues (1 mm²×15 μm,Panel (C) & (D)) removed from the tumor and adjacent normal regions.Phospholipids such as phosphatidylcholine (PC) and sphingomyelin (SM)were identified in the spectra. The peak of [PC(34:1)+K]⁺ at m/z 798 wassignificantly more intense in tumor tissue (panel C) and peaks[SM(34:1)+Na]⁺ at m/z 725, [SM(36:0)+Na]⁺ at m/z 756, and [SM(36:4)+Na]⁺at m/z 804 were significantly lower compared with normal tissue (panelD).

Example 17 On-Line Derivatization

For analysis of target analytes which have relatively low ionizationefficiencies and relatively low concentrations in mixtures,derivatization is often necessary to provide adequate sensitivity.On-line derivatization can be implemented by adding reagents into thespray solution, such as methanol/water solutions containing reagentsappropriate for targeted analytes. If the reagents to be used are stableon paper, they can also be added onto the porous material when theprobes are fabricated.

As a demonstration, 5 μL methanol containing 500 ng betaine aldehydechloride was added onto a paper triangle and allowed to dry to fabricatea sample substrate preloaded with a derivatization reagent for theanalysis of cholesterol in serum. On-line charge labeling with betainealdehyde (BA) through its reaction with hydroxyl groups has beendemonstrated previously to be very effective for the identification ofcholesterol in tissue (Wu et al., Anal Chem. 2009, 81:7618-7624). Whenthe paper triangle was used for analysis, 2 μL human serum was spottedonto the paper to form a dried spot and then analyzed by using paperspray ionization. A 10 μL ACN/CHCl₃ (1:1 v:v) solution, instead ofmethanol/water, was used for paper spray to avoid reaction between thebetaine aldehyde and methanol.

The comparison between analysis using a blank and a reagent-preloadedpaper triangle is shown in FIG. 30 panels (A) and (B). Without thederivatization reagent, cholesterol-related peaks, such as protonatedion [Chol+H]⁺ (m/z 387), water loss [Chol+H−H₂O]⁺ (m/z 369), and sodiumadduction [Chol+Na]⁺ (m/z 409), were not observed (Panel A). With thederivatization reagent, the ion [Chol+BA]⁺ was observed at m/z 488.6(Panel B). MS/MS analysis was performed for this ion and acharacteristic fragment ion m/z 369 was observed (Panel C).

Example 18 Peptide Pre-Concentration Using Modified Paper SpraySubstrate

Pre-concentration of chemicals on the paper surface using photoresisttreatment. Chromatography paper was rendered hydrophobic by treatmentwith SU-8 photoresist as described previously (Martinez et al., AngewChem Int. Ed., 2007, 46:1318-1320). Then 5 μl bradykinin 2-9 solution(100 ppm in pure H₂O) was applied on the paper surface. When thesolution was dry, the paper was put into water and washed for 10 s.After washing, the paper triangle was held in front of the MS inlet, 10μl pure MeOH was applied as solvent and the voltage was set at 4.5 kVfor paper spray. The same experiment was done with untreated papersubstrate for comparison.

FIG. 31 panel (A) shows the tandem MS spectrum of bradykinin 2-9 frompaper with photoresist treatment. The intensity of the most intensefragment ion 404 is 5.66E3. FIG. 31 panel (B) shows the tandem MSspectrum of bradykinin 2-9 from normal chromatography paper withoutphotoresist treatment. The intensity of the most intense fragment ion404 is only 1.41E1. These data show that the binding affinity betweenphotoresist-treated chromatography paper and peptide is much higher thanthat between normal chromatography paper and peptide, thus more peptidecan be kept on the paper surface after washing by water. When puremethanol is applied, these retained peptides will be desorbed anddetected by MS. This method can be used to pre-concentrate hydrophobicchemicals on the paper surface, and other hydrophilic materials (e.g.salts) can also be removed from the paper surface.

Example 19 Inverted Polarities

The polarity of the voltage applied to the probe need not match thatused in the mass analyzer. In particular, it is possible to operate theprobes of the invention with a negative potential but to record the massspectrum of the resulting positively changed ions. In negative ion mode,a large current of electrons (or solvated electrons) is produced inpaper spray. These electrons, if of suitable energy, can be captured bymolecules with appropriate electron affinities to generate radicalanions.

Alternatively, these electrons might be responsible for electronionization of the analyte to generate the radical cation oralternatively ESI might involve a solvent molecule which might thenundergo charge exchange with the analyte to again generate the radicalcation. If this process occurs with sufficient energy, characteristicfragment ions might be produced provided the radical cation is notcollisionally deactivated before fragmentation can occur.

An experiment was done on a benchtop LTP using toluene vapor, with aprobe of the invention conducted at −4.5 kV with methanol:water assolvent applied to the paper. The spectrum shown in FIG. 32 wasrecorded. One notes that ion/molecule reactions to give the protonatedmolecule, m/z 93 occur as expected at atmospheric pressure. One alsonotes however, the presence of the radical cation, m/z 92 and itscharacteristic fragments at m/z 91 and 65.

An interesting note is that the “EI” fragment ions were most easilyproduced when the source of toluene vapor was placed close to the MSinlet; i.e., in the cathodic region of the discharge between the papertip and MS inlet. This suggests that direct electron ionization byenergetic electrons in the “fall” region might be at least partlyresponsible for this behavior.

Example 20 Cartridge for Blood Analysis

FIG. 33 panel (A) shows an exemplary case for spotting blood onto porousmaterial that will be used for mass spectral analysis. The cartridge canhave a vial with a volume at the center and vials for overflows. A plug,such as a soluble membrane containing a set amount of internal standardchemical, is used to block the bottom of the vial for volume control. Adrop of blood is placed in the vial (Panel B). The volume of the bloodin the vial is controlled by flowing the extra blood into the overflowvials (Panel B). The blood in the vial is subsequently dissolved in themembrane at the bottom, mixing the internal standard chemical into theblood (Panel B). Upon dissolution of the plug, blood flows to the papersubstrate, and eventually forms a dried blood spot having a controlledamount of sample and internal standard (Panel B).

1-47. (canceled)
 48. A fluid dispenser, the dispenser comprising: afluid chamber, wherein a portion of the chamber wall comprises aninternal standard; and an outlet positioned such that fluid introducedto the chamber must interact with the portion of the chamber wall thatcomprises the internal standard prior to flowing through the outlet. 49.The dispenser according to claim 48, wherein outlet is positioned at abottom of the chamber.
 50. The dispenser according to claim 49, whereinat least portion of a bottom wall of the chamber comprises a dissolvablematerial.
 51. The dispenser according to claim 50, wherein the internalstandard coats at least a portion of the dissolvable material.
 52. Thedispenser according to claim 50, wherein the internal standardimpregnates at least a portion of the dissolvable material.
 53. Thedispenser according to claim 48, further comprising at least oneoverflow chamber.
 54. A method for dispensing a sample, the methodcomprising: providing a fluid dispenser comprising a fluid chamber,wherein a portion of the chamber wall comprises an internal standard;and an outlet positioned such that fluid introduced to the chamber mustinteract with the portion of the chamber wall that comprises theinternal standard prior to flowing through the outlet; introducing asample to the dispenser; allowing the sample to interact with theinternal standard for a time sufficient that the internal standard isintroduced into the sample; and dispensing the sample through theoutlet.
 55. The method according to claim 54, wherein outlet ispositioned at a bottom of the chamber.
 56. The method according to claim55, wherein at least portion of a bottom wall of the chamber comprises adissolvable material.
 57. The method according to claim 56, wherein theinternal standard coats at least a portion of the dissolvable material.58. The method according to claim 56, wherein the internal standardimpregnates at least a portion of the dissolvable material.
 59. Themethod according to either claim 57 or 58, wherein the sample causes theporous material to dissolve and the sample interacts with the internalstandard as the porous material dissolves.
 60. The method according toclaim 54, further comprising at least one overflow chamber.
 61. Themethod according to claim 54, wherein the sample is blood.